Avian reticuloendotheliosis virus (REV-T) is a replication-defective acute leukemia virus that transforms fibroblasts, macrophages and immature lymphoid cells. In vitro derived REV-T transformed lymphoid cells have infinite growth potential and are tumorigenic. Furthermore, these tumorigenic cells do not require the expression of any helper virus products to induce lethal reticuloendotheliosos. This is the only avian retrovirus system where it has been demonstrated that the virus can transform its target cell in vitro. REV-T contains a helper-independent sequence, designated rel, which is transcribed into a subgenomic mRNA and is presumed to be responsible for transformation. Collectively, these observations indicate that REV-T is an excellent system to study a transforming gene, its product and its activity(s) in both the in vivo target cell and alternate cell types. The specific aims of this proposal are to identify, isolate, and characterize the rel transcripts and the transforming protein of REV-T. The rel-specific mRNA from REV-T transformed cells will be isolated using cloned rel-DNA and translated in vitro. The rel protein will be purified using immunoaffinity chromatography employing tumor regressor antiserum or antibodies raised against synthetic peptides based on the established nucleotide sequence of the rel gene. The rel sequence will also be cloned into a vector which permits expression in E. coli and the protein will be identified in minicells. To confirm the transforming activity of the rel sequence, we will employ directed mutagenesis of cloned REV-T DNA coupled with transfection analysis. Directed mutagenesis will also be used to determine which regions of the rel protein are important in fibroblast, macrophage and lymphoid cell transformation. We will also define the levels and half lives of rel transcripts and protein in cells transformed by REV-T including lymphoid cells which differ in tumorigenicity. The expression of the cell homolog of rel and its protein will also be quantitated in uninfected cells. The subcellular distribution of the rel protein will be defined. Efforts will be made to associate enzymatic activities with the rel protein and to identify its potential substrates.